Lung ischemia/reperfusion injury (LIRI) is the primary cause of mortality and morbidity in patients undergoing lung transplantation. Increased reactive oxygen species (ROS) generation is a central component of LIRI. NADPH oxidase 2 (Nox2) activation leads to increased ROS generation during lung ischemia/reperfusion. New findings show that peroxiredoxin 6 (Prdx6) is required for Nox2 activation. Prdx6 is a bifunctional protein that expresses peroxidase and phospholipase A2 (PLA2) activities. While the peroxidase activity scavenges ROS, the PLA2 activity activates Nox2 during agonist stimulation and ischemia/reperfusion in lung endothelium, alveolar macrophages and polymorphonuclear neutrophils (PMN). Although the requirement of Prdx6PLA2 for Nox2 activation is established, the mechanism of such activation remains elusive. The main products of Prdx6PLA2 are a lysophospholipid and a free fatty acid. Our preliminary studies show that lysophosphatidic acid (LPA) receptor blockade decreases Nox2-dependent ROS generation during agonist stimulation in pulmonary microvascular endothelial cells (PMVEC) and isolated perfused lungs, and that LPA treatment recovers ROS generation in PMVEC from Prdx6 null mice. Therefore, we will study the role of Prdx6PLA2- derived LPA signaling in Nox2 activation during lung ischemia/reperfusion and the contributions of LPA signaling to LIRI. Our central hypothesis is that blocking LPA signaling ameliorates LIRI. This hypothesis will be addressed through two specific aims. Aim 1 will evaluate the requirement of Prdx6-derived LPA signaling for Nox2 activation during ischemia/reperfusion using an in vitro model of flow-adapted PMVEC. Activation will be determined by the translocation of cytosolic components of Nox2 to the plasma membrane and ROS generation. Aim 2 will evaluate the contributions of LPA signaling to LIRI using in situ and in vivo murine models. ROS generation, lipid peroxidation, inflammation, apoptosis, neutrophil infiltration and edema will be measured as indicators of LIRI. The use of an in vitro model, knockin and knockout cells, pharmacological inhibitors and shRNA technology will allow us to elucidate the mechanism of LPA-driven ROS generation during ischemia/reperfusion. The use of isolated perfused lungs will help understanding the dynamics of LPA- driven intravascular ROS generation and oxidative damage, and the contributions of LPA signaling to lung ischemia/reperfusion. The use of an in vivo hilar clamp murine model will give a global perspective that could shade light on the contributions of other cells (PMN, macrophages) to LIRI, and the potential therapeutic value of LPA receptor blockers in the prevention of LIRI. Thus, the proposed work will provide valuable new information related to Nox2 activation during lung ischemia/reperfusion and therefore can offer an opportunity to influence the activation of the oxidase. Understanding the regulation of Nox2 activation will provide important insights for our understanding of the mechanisms for LIRI and could open the door to pharmacologic intervention in order to limit Nox2-driven ROS generation during LIRI.